*3.2. The Parameters Chromosome Size, S-Phase Duration, and Replication Rate Allow Estimating the MO per Chromosome in Any Organism*

In a recent study, our group developed a formula able to estimate the MO required to duplicate an entire chromosome within the S-phase duration [8]. The development of this formula was based on the bidirectional movement of the replication forks, replication rate, S-phase duration, and the chromosome size in question. Although used only in *T. brucei* so far, this formula can be applied in any cell type. To demonstrate this, we estimated the MO in *T. cruzi*, *L. major*, *T. brucei* (using updated parameters), *S. cerevisiae*, and *S. pombe*, as shown in Table 1.


**Table 1.** Calculation of the minimum number of origins (MO) per chromosome in trypanosomatids (*T. cruzi*, *L. major*, and *T. brucei*) and yeasts (*S. cerevisiae* and *S. pombe*).

<sup>1</sup> *T. cruzi*: S-phase duration = 591.6 min (current study), replication rate = 2.05 kb·min−<sup>1</sup> [23]; <sup>2</sup> *L. major*: S-phase duration = 192 min (current study), replication rate = 2.44 kb·min−<sup>1</sup> [3]; <sup>3</sup> *T. brucei*: S-phase duration = 138.6 min [8], replication rate = 3.06 kb·min−<sup>1</sup> [8]; <sup>4</sup> *S. cerevisiae*: S-phase duration = 30 min [34,35], replication rate = 1.6 kb·min−<sup>1</sup> [11]; <sup>5</sup> *S. pombe*: mitotic S-phase duration = 24 min [25,26], mitotic replication rate = 0.91 kb·min−<sup>1</sup> [24].

Among the single-celled eukaryotes analyzed here, *T. cruzi* draws attention because it is the only organism that requires only one origin per chromosome (MO = 1) to replicate its nuclear genome within the S-phase duration, as displayed in Table 1. *L. major*, on the other hand, requires more than one origin per chromosome to replicate its larger chromosomes (>1000 kb), while *T. brucei*, *S. cerevisiae*, and *S. pombe* requires more than one origin per chromosome to replicate their nuclear genomes, even for small chromosomes (<1000 kb), as shown in Table 1. As the formula to estimate the MO (Equation (3)) depends on the chromosome size, S-phase duration, and replication rate, the explanation for these

organisms possess different MOs is related to these variables. For instance, *T. cruzi* has a long S-phase duration (9.86 h or 0.411 ccu) relative to other organisms analyzed here shown in Figure 1B, which justifies its MO per chromosome equaling 1, as presented in Table 1. Figure 1B and Table 1 show that *T. brucei* has an S-phase duration and replication rate similar to *L. major*; however, its chromosomes are larger than 1,000,000 bp (called megabase chromosomes [36]), which justifies the use of more than one origin per chromosome. *S. cerevisiae*, on the other hand, has a short S-phase duration (0.52 h or 0.347 ccu) and a low replication rate (1.6 kb·min−<sup>1</sup> [11]), which imply, according to our formula, high MO values, as shown in Figure 1B and Table 1. *S. pombe*, in turn, has longer chromosomes, a short S-phase duration (0.4 h or 0.1 ccu) and a low replication rate (0.91 kb·min−<sup>1</sup> [24]), which also imply high MO values. These data are also shown in Figure 1B and Table 1.

It is difficult to establish a reason why some organisms need a different number of origins during the S-phase. However, we can speculate that the number of origins needed to replicate all chromosomes during the S-phase is closely related to the S-phase duration itself. The question that remains is as follows: does the number of fired origins determine the S-phase duration, or is the S-phase duration robust, and a different number of origins is required to maintain this robustness? Although some studies point to robustness in S-phase duration [6–8], further studies are necessary to confirm which of these questions is the correct one.

It is worth to mention that among the parameters used to determine the MO, the replication rate is the most prone to alterations. Many factors can change the replication rate, such as decreased nucleotide pool [37,38], replication-transcription conflicts [39,40], DNA damage [41,42], among others, all of which leads mostly to some replication stress [43]. In other words, replication stress can be defined, in general, as the slowing of replication rate [44]. Thus, cells under replication stress probably would show different MO values relative to those estimated using the average replication rate from a wild type population.
